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Detection kit and detection method for detecting animal C-reactive protein based on amide group nanometer latex enhanced turbidimetry

A detection kit and nano-latex technology, which is applied in the field of medical immunodiagnosis, can solve problems such as the absence of C-reactive protein, and achieve the effects of good specificity, high sensitivity, and rapid response

Active Publication Date: 2015-11-18
SHENZHEN HUISONG TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no detection reagent for animal C-reactive protein in latex-enhanced immunoturbidimetric method on the market

Method used

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  • Detection kit and detection method for detecting animal C-reactive protein based on amide group nanometer latex enhanced turbidimetry
  • Detection kit and detection method for detecting animal C-reactive protein based on amide group nanometer latex enhanced turbidimetry

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Experimental program
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Embodiment approach

[0028] As another preferred embodiment of the present invention, the nanolatex in the C-reactive protein antibody cross-linked amide group nanolatex is covalently bonded to the amino group or carboxyl group of the C-reactive protein antibody through the amide group on the surface. In this preferred case, the combination of the amide group nanolatex and the antibody is through the covalent bonding of the amide group on its surface to the amino group or carboxyl group of the antibody, and there is a bridging chemical arm between the microsphere and the antibody, which reduces the position. The blocking effect not only improves the binding rate of the antibody, but also provides a suitable three-dimensional structure for the antibody, effectively protecting the active area where the antibody binds to the antigen, and improving the sensitivity of the reaction.

[0029] The present invention also provides a method for detecting animal C-reactive protein based on amide group nano-lat...

Embodiment 1

[0040] 1. Preparation of detection kit:

[0041] 1. Take 1ml of polystyrene latex particles (particle diameter: 80nm) with amide groups on the surface and dilute to 100ml with 10mM 2-morpholineethanesulfonic acid buffer, pH=5.0, and the final concentration is 1%.

[0042] 2. Add 0.1% w / v 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.1% w / v N-hydroxysuccinamide to activate the latex, and bathe in water at 37°C for 1 hour .

[0043] 3. Centrifuge, remove the supernatant (containing the residual activated cross-linking agent), then add 2 mg of CRP polyclonal antibody for cross-linking reaction, and bathe in water at 37°C for 4 hours.

[0044] 4. Finally, add 1ml of latex blocking solution containing 2% w / v protein BSA, 0.1% w / v sodium azide and 10mmol / L phosphate buffer at pH=7.0 to block the residual activation sites on the latex surface to obtain R2 reagent.

[0045] 5. Take 10ml of 10mmol / L phosphate buffer solution with pH=7.0, add 1%w / v PEG20000, 1%w / v ...

Embodiment 2

[0056] 1. Preparation of detection kit:

[0057] 1. Take 1ml of polystyrene latex particles (particle diameter: 200nm) with amide groups on the surface and dilute to 200ml with 5mM 2-morpholineethanesulfonic acid buffer solution with pH=6.0, the final concentration is 0.5%.

[0058] 2. Add 0.05% w / v 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.05% w / v N-hydroxysuccinamide to activate the latex, and bathe in water at 37°C for 1 hour .

[0059] 3. Centrifuge, remove the supernatant (containing the residual activated cross-linking agent), then add 2 mg of CRP polyclonal antibody for cross-linking reaction, and bathe in 37°C water for 3 hours.

[0060] 4. Finally, add 1ml of latex blocking solution containing 1%w / v trehalose, 0.05%w / vProclin-300 and 20mmol / L citric acid-phosphate buffer at pH=8.0 to block the residual activation sites on the latex surface. The R2 reagent is obtained.

[0061] 5. Take 10ml of 20mmol / L phosphate buffer solution with pH=8.0, ad...

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Abstract

The invention provides a detection kit and detection method for detecting animal C-reactive protein based on amide group nanometer latex enhanced turbidimetry. The detection kit comprises R1 reagent and R2 reagent. The R1 reagent comprises 10 to 50 mmol / L of first buffer solution, 0.2% to 2.5% w / v of coagulant, 0.1 to 2% w / v of surfactant and 0.01% to 0.1% w / v of first preservative. The R2 reagent comprises 0.5% to 2% w / v of C-labbeled reactive protein antibody crosslinking amide group nanometer latex, 0.2 to 2% w / v of stabilizer, 10 to 50 mmol / L of second buffer solution and 0.01 to 0.1 % w / v of second preservative. The detection kit and the detection method have the advantages of being simple to operate, quick to react, high in sensitivity and good in specificity.

Description

technical field [0001] The invention relates to the field of medical immunodiagnosis, in particular to a detection kit and a detection method for animal C-reactive protein based on amide group nano-latex enhanced turbidimetry. Background technique [0002] C-reactive protein (C-reactive protein, referred to as CRP) was first discovered by Tillet and Francis in 1930, and it was named because it can react with the C polysaccharide of the cell wall of Streptococcus pneumoniae. As an acute phase response protein, CRP is mainly produced by the liver under the mediation of interleukin-6, and at the same time regulated and stimulated by interleukin-1 and tumor necrosis factor α, etc., in atherosclerosis, kidney, CRP is also synthesized in neurons and vacuolar macrophages. In animals, CRP also exists. For example, it has been found in dogs, pigs, rabbits, horseshoe crabs, and river mussels. Dog CRP is a ring-shaped pentraxin with a molecular weight of about 100kD and composed of fi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/82G01N33/68
Inventor 陈海彬唐金春高燕静
Owner SHENZHEN HUISONG TECH DEV
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